(1) Field of the Invention
This invention relates to the prevention of dental caries, and more particularly to the immunization against caries employing the glucosyl transferase (dextransucrase) enzyme (GTF) involved in the synthesis of the water-insoluble extracellular polysaccharide from sucrose by the action of cariogenic bacteria. More specifically, it relates to methods of obtaining more highly purified glucosyl transferase enzyme. It further relates to cross protection via immunization with serotype c GTF, or with serotype g CTF or with serotype a GTF.
(2) Description of the Prior Art
Dental caries is the most prevalent infectious disease in the western world, and the cost of its treatment exceeds that of any single bacterial infection.
Studies have indicated that the formation of carious lesions on teeth is related to the interaction between carbohydrates (notably sucrose) in the diet and specific bacteria on tooth surfaces. The cariogenic bacteria, predominantly streptococci, adhere to the surfaces of teeth by synthesizing extracellular polysaccharides from sucrose. Streptococcus mutans is the principle organism associated with the dental caries in humans and will also produce disease in rodents and in primate models. O'Brien, T. C. 1976. Introduction and rationale for continued investigations on a vaccine as an approach to dental caries prevention. Immunology Abstracts (Special Issue):3. Gibbons, R. J. and J. van Houte. 1975. Bacterial adherence in oral microbial ecology. Ann. Rev. Microbiol. 29:19. These organisms ferment carbohydrate moieties which results in the production of acid leading to demineralization of the tooth enamel.
These polysaccharides, which are generally either polyglucans or polyfructans (levan), "glue" the bacterial cells together and help them adhere to the teeth. The polysaccharides thus promote action between the bacteria and further sucrose ingested by the host animal and thereby facilitate the formation of further polysaccharides. Moreover, the extracellular polysaccharides thus produced are believed to play significant roles in plaque formation and in the consequent development of caries. The polysaccaride polymers are synthesized from sucrose by a group of extracellular and cell-associated consitutive enzymes collectively called glucosyltransferase (GTF).
At least two types of GTF enzymes have been described on the basis of the product synthesized. One type of GTF enzyme(s) will synthesize predominantly water-insoluble glucose polymers (called mutan). Guggenheim, B. 1970. Enzymatic hydrolysis and structure of water-insoluble glucan produced by glucosyltransferases from a strain of Streptococcus mutans. Helv. Odontol. Acta 14:89. A second type of GTF enzyme(s) is primarily responsible for the synthesis of water-soluble glucose polymers, which consist predominantly of .alpha.1-6 linkages. Mutan, containing significant additional .alpha.1-3 linkages appears to be important in the adherence phenomena of S. mutans. Mukasa, H., and H. D. Slade. 1973. Mechanisms of adherence of S. mutans to smooth surfaces. I. Roles of insoluble dextran-levan synthetase enzymes and cell wall polysaccaride antigens in plaque formation. Infect. Immun. 8:555.
S. mutans cells have been used in experiments designed to study the effects of immunization on experimental dental caries in rodent and primate model systems. Taubman, M. A. 1973. Role of immunization in dental disease. In Comparative Immunology of the Oral Cavity. Edited by S. Mergenhagen and H. Scherp. U.S. Government Printing Office, Washington, D. C. P. 138. Taubman, M. A., and D. J. Smith. 1974. Effects of local immunization with Streptococcus mutans on induction of salivary IgA antibody and experimental dental caries in rats. Infect. Immun. 9:1079. McGhee, J. R., S. M. Michalek, J. Webb, J. M. Navia, A. F. R. Rahman, and D. W. Legler. 1975. Effective immunity to dental caries: protection of gnotobiotic rats by local immunization with Streptococcus mutans. J. Immunol. 114:300. Bowen, W. H., B. Cohen, M. F. Cole, and G. Colman. 1975. Immunization against dental caries. British Dent. J. 139:45. Lehner, T., S. J. Challacombe, and J. Caldwell. 1975. Immunological and bacteriological basis for vaccination against dental caries in Rhesus monkeys. Nature 254:517. Evans, R. T., F. G. Emmings, and R. J. Genco. 1975. Prevention of Streptococcus mutans infection of tooth surfaces by salivary antibody in irus monkeys; (Macaca fasicularis). Infect. Immun. 12:293. In these experiments the use of whole cell antigens, which often bear GTF on the surface, has resulted in diminished colonization of S. mutans or reduced dental caries formation. Immunized animals often contained demonstrable serum and/or salivary antibody to GTF in addition to antibody of other specificities. Emmings, F. G., R. T. Evans; and R. J. Genco. 1975. Antibody response in the parotid fluid and serum of irus monkeys (Macaca fasicularis) after local immunization with Streptococcus mutans. Infect. Immun. 12:281; Russell, M. W., S. J. Challacombe, and T. Lehner. 1976. Serum glucosyltransferase-inhibiting antibodies and dental caries in Rhesus monkeys immunized against Streptococcus mutans. Immunology 30:619; Genco, R. J., R. T. Evans, and M. A. Taubman. 1974. Specifically of antibodies to Streptococcus mutans; significance in inhibition of adherence. Adv. Exp. Med. Biol. 45:327.
The use of materials containing GTF enzymatic activity as antigens has, at times, also resulted in caries reductions in preliminary experiments using the rodent model. Hayashi, J. A., I. L. Shklair, and A. N. Bahn. 1972. Immunization with dextransucrases and glycosidic hydrolases. J. Dent. Res. 51:436. However, similar experiments in primates have seldom shown reductions. Lehner, T., S. J. Challacombe, and J. Caldwell. 1975. An immunological investigation into the prevention of caries in deciduous teeth of Rhesus monkeys. Arch. Oral Biol. 20:305.
In vitro experiments have demonstrated that antibody to GTF can interfere with the formation of the polysaccharide product as well as reduce the adherence of S. mutans to hard surfaces such as wire or glass. Fukui, K. Y. Fukui, and T. Moriyama. 1974. Some immunochemical properties of dextransucrase and invertase from Streptococcus mutans. Infect. Immun. 10:985. Therefore, the significance of GTF in the manifestation of S. mutans virulence, the ability to immunologically inhibit GTF enzyme function, and the presence of antibody directed to GTF in animals protected against caries, implicated these enzymes as potential antigens for the study of the effects of immunization on experimental dental caries.
A number of vaccines have been proposed for immunization against dental caries in animals. Various of these proposals are summarized in U.S. Pat. No. 3,879,545. The invention claimed in that patent relates to caries-preventive vaccines incorporating as the active ingredient thereof a polyfructan (or levan) polysaccharide produced by elaboration of certain strains of streptococcus, particularly Streptococcus Strain SS2. The immunization technique, according to the patentee, has been found to result in the formation of antibodies against the heterogeneous micro-organisms in the recticuloendothelial system and in the blood, and to result in significant decreases in the formation of caries lesions in host animals subjected to innoculation with such organisms. However, the patentees also disclose investigating the use of dextransucrase enzyme for immunization against caries formation.
As disclosed in U.S. Pat. No. 3,879,545, the patentees had believed that since the enzymes dextransucrase and levansucrase are produced by S. mutans and S. Strain SS2, respectively, and are involved in the synthesis of dextran and levan, and these polysaccharides are believed to produce caries, that immunization with these enzymes might result in the production of antibodies to the enzymes which would neutralize their activity in vivo, thus inhibiting synthesis of the polysaccharides and resulting in decreased plaque formations and lowered incidence of caries. The patentees disclose, however, that their experimentations failed to confirm this hypothesis. Nevertheless, the data disclosed did appear to show that while enzyme preparations purified as described by Guggenheim and Newbrun in Helv. Odontol. Acta. 13:84-97(1969) were ineffective in two routes of systemic immunization, a more crude form was somewhat effective against infectious inoculations with S. mutans 6715.
In U.S. Pat. No. 3,931,398 there is disclosed a locally administered vaccine containing dextransucrase (glucosyl transferase). As disclosed in the patent, this somewhat impure enzyme was prepared by the method of Guggenheim and Newbrun, supra, from the supernatant liquid of an 18-hour culture of S. mutans 6715 grown on 8% sucrose. These preparations, as disclosed in the patent, had 10 units of dextransucrase activity per mg. of protein, one unit of the enzyme being defined as the amount required to catalyze transformation of 1 mg. of sucrose to dextran in 1 hour (releasing 0.52 mg. of fructose) at pH 6.8. The activity was measured by determining the amount of released reducing sugars. While the patentee speculates that the use of purer forms of the dextransucrase enzyme would result in further diminution of the mean caries scores disclosed, no method is disclosed in this patent of obtaining a purer form of the enzyme.